Mechanisms of Eukaryotic Transcription Initiation

Abstract

Regulation of gene expression determines cellular phenotype and when disrupted can lead to cancer and other disease states. For many genes regulation occurs via control of transcription initiation. Eukaryotic transcription initiation of mRNA is a complex process wherein a set of general transcription factors and RNA polymerase II (Pol II) assemble into a pre-initiation complex (PIC) at a promoter. The protein factors involved and the physical interactions between them and the promoter have been the subject of many years of research in the field. However, the topological dynamics of the DNA in eukaryotic PICs are not well understood, limiting mechanistic understanding of promoter unwinding, transcription start-site scanning, and promoter escape.Using magnetic tweezers we have observed ATP-dependent promoter opening by single minimal PICs comprised of TBP, TFIIB, TFIIE, TFIIF and TFIIH and Pol II assembled on the TATA-dependent HIS4 promoter from Saccharomyces cerevisiae. Single molecule traces of TFIIH-dependent promoter opening reveal that the size of the transcription bubble is dynamic during initiation, displaying multiple transitions between intermediates with varying extents of open DNA. Furthermore, in contrast to prokaryotic initiation, promoter opening does not appear to be accompanied by significant DNA compaction. These observations place constraints on the promoter opening and transcription start site scanning mechanisms for yeast PICs.